Conformational changes and loose packing promote E. coli Tryptophanase cold lability
© Kogan et al; licensee BioMed Central Ltd. 2009
Received: 7 January 2009
Accepted: 8 October 2009
Published: 8 October 2009
Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.
We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25°C and even further upon cooling to 2°C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9Å resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study.
We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.
Enzymes can undergo a reversible loss of activity at low temperatures, a process that is termed cold inactivation [1, 2]. This phenomenon is found in a widespread variety of oligomeric enzymes, e.g, pyrophosphatase , pyruvate carboxylase , alcohol dehydrogenase , as well as PLP-dependent enzymes such as glutamic acid decarboxylase  and tryptophanase (Trpase) [7–9]. It has been proposed that cooling evokes major changes in hydrophobic interactions which leads to destabilization of the enzyme quaternary structure concomitant with dissociation into its corresponding subunits . These changes can in most cases be reversed after re-warming [11, 12]. Sometimes, however, cold-induced dissociation is followed by an aggregation step, which causes the irreversibility of the process [13–16]. It is believed that at low temperatures the entropic term for association becomes less favorable since the solvent molecules released upon association are more ordered. In general, the models for cold inactivation predict that the pivotal role in cold inactivation is played by hydrophobic interactions , but direct experimental evidence is lacking.
E. coli tryptophanase (tryptophan indole-lyase, Trpase, EC 220.127.116.11) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyses α,β-elimination and β-replacement reactions of L-tryptophan. The molecular weight of Trpase from different species ranges from 200 to 220 kD. Trpase consists of four identical subunits (~52 kD per monomer), each of which covalently binds one molecule of PLP. The tetramer possesses a D2 symmetry, therefore it is a dimer of dimers with two different dimeric interfaces: a catalytic interface and a non-catalytic interface (Figure 1) [18–26].
The crystal structure of holo Trpase isolated from Proteus vulgaris  and of another two crystal structures of E. coli Trpases in their apo form were determined [28, 29]. Both crystal structures of apo E. coli Trpases share the same fold as the holo P. vulgaris Trpase, but structural alignment of the three crystal structures revealed significant differences in the relative orientation of the two domains of the enzyme . We compared these known crystal structures to the two new crystal structures of Trpase mutants we determined in order to gain structural insights into the cold-induced conformational changes and PLP release. In addition, we sought structural themes to account for the stability of E. coli dimers which do not dissociate into monomers as do those from P. vulgaris.
Enzymatic activity measurements
Specific activity of wt-Trpase and its mutants at 25°C and after cooling to 2°C in Tricine -KCl buffer, pH = 7.5.
45.7 ± 2.5
29.7 ± 1.7
42.8 ± 3.2
5.0 ± 1.4
36.9 ± 2.9
3.7 ± 0.8
0.2 ± 0.05
0.08 ± 0.03
A summary of physical properties of residues affecting cold lability.
On the catalytic interface
Involved in hydrogen bonding with PLP and Arg103 from adjacent subunit
On the catalytic interface
On the non-catalytic interface
Close to the catalytic interface
Oxidized with 2-mercaptoethanol
Close to the catalytic interface
On the non-catalytic interface
Involved in inter subunit β-sheet network
On the non-catalytic interface
Involved in inter subunit β-sheet network
On the non-catalytic interface
Involved in inter subunit β-sheet network
In the Y74F mutant, the observed loss of activity at 25°C is presumably attributed to the proximity of residue 74 to the substrate binding site . In the wt Trpase the hydroxyl group of Tyr74 is hydrogen bonded to both Arg103 (at a 2.9 Å distance), and to the phosphate group of PLP (of the neighbouring subunit in the catalytic dimer) through a bridging water molecule. Replacing Y74 by phenylalanine eliminated the hydrogen bonds of the hydroxyl group thereby reducing its affinity towards PLP. Hence, the Y74F mutant exhibited negligible activity compared to the other Trpases.
Time dependence profile of cold inactivation and dissociation
The cold dissociation time profiles revealed the same pattern, with the following initial rate constants: (3.2 ± 0.1) × 10-3 min-1 (0.84 ± 0.1) × 10-3 min-1 and (0.42 ± 0.1) × 10-3 min-1 for C298S, W330F and wt Trpase, respectively (Figure 2B). The rate constants for cold inactivation and dissociation are similar for the wt and the W330F mutant, but in the case of C298S there is a factor of ~4 between them. This may be attributed to the relative proximity of residue 298 to the PLP and substrate binding sites as well as to its location on the catalytic interface, in contrast to that of the 330 site. The PLP in the C298S mutant is loosely bound allowing, relative to W330F and the wt, a quicker cold inactivation and dissociation.
Dissociation of E. coli Trpases to dimers
The effect of cooling on the dissociation of wt Trpase and its mutants to dimers.
Degree of dissociation to dimers (%)
E. coli Trpase
20 ± 1
64 ± 1
61 ± 2
17 ± 3
66 ± 4
70 ± 5
87 ± 3
88 ± 1
69 ± 4
90 ± 1
80 ± 1
83 ± 2
In contrast to the holo forms, all apo forms of Trpase markedly dissociate to dimers, about 70% at 25°C and about 90% upon cooling to 2°C suggesting that PLP has a major effect on the stability of the holo form. Once the PLP molecule is removed, the differences in the extent of dissociation among the wt and the mutants are very small. However, in the holo forms in which PLP is present, the differences between the mutants stem primarily from the effect of mutation, which in all three mutants presumably cause a reduced fit between the dimers relative to the wt form. Thus, the apo form of all Trpases showed a similar lack of stability towards cooling, indicating that the expulsion of PLP renders the apo form equally susceptible to a very pronounced cold driven dissociation.
High pressure studies
Hydrostatic pressure causes unfolding of proteins because there is a decrease in the volume of the protein-solvent system upon unfolding . Generally, three factors are considered to contribute to ΔVo: electrostriction of charged and polar groups that became exposed to solvent upon unfolding or conformational change; collapse of packing defects and internal cavities; volume effects transfer of hydrophobic groups from the protein interior to water. The two first factors provide a negative contribution to ΔVo. However, the third factor, the exposure of hydrophobic groups to solvent can give both negative and positive contributions for changes in hydration volumes, depending on pressure and temperature .
In the case of the wt Trpase, the conformational change from a closed to an open conformation under increasing pressure is accompanied by large negative value of ΔVo. We used the Voronya  tool for analyzing the packing of the two crystal structures of wt Trpase which are found in the different conformations (for closed conformation we used pdb entry 2C44; and for the open conformation pdb entry 2OQX). The cavities analysis did not reveal any significant difference between the two conformations. Therefore, the collapse of internal cavities can not be the reason for the large negative value of ΔVo. Presumably, the main attributing factor to the large decrease in the volume is hydration of both polar and hydrophobic groups in the catalytic interface, which became exposed to water in the open conformation. At moderate pressures, the unfolding is reversible, but at pressures above 2.5 Kbar, subunit dissociation and irreversible denaturation are frequently observed.
The overall crystal structure of Y74F and C298S
Data collection and refinement statistics* for Y74F and C298S mutants of E. coli Trpase.
a = 118.7Å
a = 120.5Å
b = 120.2Å
b = 118.8Å
c = 171.7Å
c = 171.5Å
α = 90°
α = 90°
β = 90°
β = 90°
γ = 90°
γ = 90°
Highest resolution shell
2. 05-1.98 Å
Total number of measured reflections
Total number of independent reflections
Resolution range (Å)
RMS deviations from restraints target value
Bond length (Å)
Bond Angle (°)
Average B factor
(for protein atoms) (Å2)
Dissociation of P. vulgaris Trpase
The effects of cooling and reactivation on the degree of dissociation of apo wt Trpase from E. coli and P. vulgaris
Degree of dissociation (%)
P. vulgaris Trpase
37.3 ± 1.2
44.5 ± 3.4
18.2 ± 4.0
P. vulgaris Trpase
2°C, 24 h
21.7 ± 0.6
45.4 ± 0.7
32.9 ± 1.6
P. vulgaris Trpase +1 mM PLP
25°C, 2 h
92.6 ± 0.8
7.4 ± 0.2
E. coli Trpase
30.0 ± 2.2
70.0 ± 5.0
E. coli Trpase + 1 mM PLP
25°C, 1 h
100 ± 0.6
Analysis of the non-catalytic interface in both Trpases (from E. coli and P. vulgaris) revealed that the β-sheet located at the core of the tetramer is not exposed to the solvent. Furthermore, the four β-strands are structurally similar, but differ in their amino acid content. Met57, Met13 and Val14 of P. vulgaris Trpase are replaced by Val59 and Val15 and Ile16 in the E. coli Trpase (Table 2). The two valine residues and the isoleucine residue of E. coli Trpase are more hydrophobic (see table 2) than the corresponding two methionine residues and the valine residue of P. vulgaris . Thus, the presence of the more hydrophobic core in the region of the non-catalytic interface of E. coli presumably leads to a higher stability of the dimeric structure of apo Trpase from E. coli compared to Trpase of P. vulgaris.
At low temperatures and low concentrations E. coli Trpase undergoes a reversible loss of activity, followed by dissociation into dimers. Complete dissociation/inactivation occurred after incubation for 20 h at 2°C and association/reactivation occurred upon warming for few hours. This was previously reported in a detailed time course study for both the wt Trpase and its W330F mutant [11, 12, 27]. To further explore the temperature driven changes in the hydrophobic interactions at the non-catalytic or at the catalytic interfaces of E. coli Trpase we examined the role of single point mutation at each interface on enzyme activity, dissociation and crystal structure.
where TE-PLPclose is the holo tetrameric active Trpase at 25°C in a close conformation; TE-PLPopen is the holo tetrameric enzyme in the open conformation at 2°C; TE... PLP is the non-covalently bound complex in an open conformation and TE is the apo enzyme in an open conformation at 2°C. Step 4 is the dissociation of the tetrameric form to dimers, TE to DE.
Cooling (step 1) weakens the hydrophobic interactions resulting in a conformational change and a corresponding modified solvation. The change from the closed to an open conformation is associated with a reduction in the tight packing of the tetramer and is enhanced by the point mutations. This conformational change is further supported by the present high pressure studies showing that increasing hydrostatic pressure has the same effect as decreasing temperature in affecting the conformational equilibrium.
The conformational change at low temperatures results in breaking the covalent aldimine bond between residue Lys270 (E. coli numbering) and the PLP molecule (step 2). This overcomes the rest of the non-covalent binding interactions between PLP and Trpase, and results in the separation of the PLP from the active site (step 3) leading to dissociation into dimers (step 4). This suggested sequence for PLP-Trpase interactions is further supported by HPLC-Diode array profiles which could not detect PLP-bound dimers.
Our results suggest that even minor changes in the size of the amino acid side chain reduce the tight fit that is presumably required for the stability of the holo tetrameric enzyme. The reduced fit due to mutations along the two symmetry axis resulted in a higher vulnerability to cooling, which enhances the dissociation and the release of PLP. Changes in side chain hydrophobicity in the present mutants were found to be less relevant to the stability of the tetramer.
We also compared the dissociation of the wt and the three mutants in their apo form and found that their dissociation into dimers occurs even at 25°C to the same extent (Table 3). Upon cooling of the apo forms to 2°C, an additional dissociation of about 15% was observed. The effect of the point mutation on the cold dissociation was observed only in the holo form. We found that at 2°C, a factor of 3 in the extent of dissociation was observed between the wt and its mutated holo forms (Table 3). The loss of PLP overshadows the relative small contributions of the different interactions in the mutants, suggesting that these interactions at both interfaces have a relatively minor effect on the cold dissociation of E. coli Trpase.
In contrast to the similarity found in the degree of cold inactivation, dissociation (Tables 1&3) and the crystal structures (Figure 5), differences were observed in the kinetics of cold inactivation and dissociation between the mutants and the wt Trpase (Figure 2). The location of the point mutation, i.e. on the catalytic (C298S) vs. the non-catalytic (W330F) axis, affects primarily the kinetics of cold lability. Notably, while a qualitative correlation was observed between the kinetics of cold inactivation and dissociation, it does not follow a quantitative relationship. While cold inactivation is a direct result of primarily the enamine PLP-Lys270 scission, the following collapse of the quaternary structure into dimers results from numerous protein-protein and protein-solvent interactions including hydrophobic, electrostatic and hydrogen bonds. A detailed description of this multi-step process requires the study of additional point mutations.
To summarize, the observed cold lability of E. coli Trpase results from the release of PLP and from the preclusion of the tight assembly of the tetramer. The wild-type system is balanced to favor the tetramer stabilization, and even a small perturbation of the interaction energy causes it to shift toward dimers.
The cold inactivation and dissociation of holo E. coli Trpase is mainly affected by the release of the cofactor PLP. In the absence of PLP variable packing-interactions on the monomers' interface due to the mutation (Y74F, C298S and W330F) make additional, relatively small contribution to the cold dissociation of E. coli Trpase to dimers. The hydrophobic interactions along the non-catalytic interface may account for the difference in dissociation into dimers vs. monomers in E. coli and P. vulgaris Trpases.
S-(o-nitrophenyl)-L-cysteine (SOPC) was synthesized as described previously . PLP, Tricine, L-tryptophan, 2-mercaptoethanol, protamine sulfate, ampicillin, buffers and (NH4)2SO4 were purchased from Sigma. Other chemicals obtained from various commercial suppliers were of pure or extra pure grade.
Wild-type Trpase was isolated from E. coli SVS 370 cells containing the tnaA gene on plasmids by a procedure described previously . Additional purification was achieved by anion exchange chromatography on DEAE-Sephadex A-50 or DEAE-Sepharose 4B-CL, and size exclusion chromatography on Sephacryl S-300-HR [11, 45]. The protein fractions with the highest specific activity and homogeneity in SDS polyacrylamide gel electrophoresis (greater than 97% purity) were combined and concentrated by total precipitation with (NH4)2SO4 or in an Amicon ultrafiltration YM-30 membrane and frozen in 0.3-ml aliquots at -80°C. W330F and C298S mutants of E. coli Trpase were isolated in the same way from E. coli SVS 370 cells containing the tnaA gene with the respective site-directed mutation. The Y74F mutant was expressed under lac., on a pET17b plasmid in BL21(DE3) cells. Wild-type Trpase from P. vulgaris was isolated from E. coli SVS 370 cells containing the tryptophanase gene from P. vulgaris in the pAVK plasmid . Protein concentration during purification was determined using the Bradford reagent  with BSA as a standard. The concentration of each purified enzyme (wt, C298S and Y74F) was determined at 278 nm, taking the absorbance A1% values of 9.19 for holo P. vulgaris and holo E. coli and 7.95 for apo wt-Trpases . The concentration of W330F was determined using the value of 7.64 for the holo form . The apo enzymes were prepared by overnight dialysis of the holo enzymes against 0.1 M sodium phosphate-ethylenediamine buffer, pH 8.0, at 4°C.
Enzymatic activity measurements
Activity was measured by a spectrophotometric method with the chromogenic substrate analog, S-(o-nitrophenyl)-L-cysteine (SOPC), as described in Suelter et al., [21, 48] using the 8453A Hewlett Packard spectrophotometer connected to a UC-F-10 Julabo thermostated bath (JTB) (± 0.1°C). A-10 μl enzyme aliquots (0.3-0.5 mg protein/ml, specific activity 42-50 μmol min-1mg protein-1) were stirred into 1 ml (final volume) of 0.6 mM SOPC in 50 mM potassium phosphate buffer, pH 8.0, at 25°C and. Initial activity was measured for 1 min by following the decline in OD370 nm (Δε = -1.86 × 103 M-1cm-1). One unit of Trpase is defined as the amount required for the decomposition of 1 μmol SOPC to the product o-nitrothiophenolate in 1 min at 25°C. The activity measurements related to cold inactivation were carried out as described above. The activities of the Trpases at 2°C were measured within 30 to 60 sec, a time interval which is short enough to prevent reversibility of the cold-inactivated form. We previously have shown that the reactivation requires a minimum of 1 h of re-warming to 25°C [11, 12, 27, 49]. The concentrations of the wt Trpase and of W330F, C298S mutants were 0.3-0.5 mg/ml, while the concentration of the Y74F mutant was 10-20 mg/ml .
Time dependence of cold inactivation and dissociation
The time dependence of cold inactivation of wt Trpase and its mutants were performed at 25°C in 50 mM Tricine-KOH buffer at pH 7.5 containing 100 mM KCl (Tricine-KCl), 5 mM 2-mercaptoethanol, 50 μM PLP. The enzyme solution (0.5-1 ml) was kept on ice and slightly stirred. Aliquots of 10-20 μl were removed at various times for assay at 25°C. The time profile of cold dissociation for the wt, C298S and W330F was followed by HPLC analysis as described below.
HPLC measurements of dissociation degree for holo and apo Trpases
HPLC analysis of Trpases was performed at 25°C and 2°C with a Waters 99 supplied Photodiode Array detector UV using a gel filtration column BIOSEP-SEC S-3000 (7.5 × 600 mm), equipped with a water jacket connected to a thermostated bath (UC-F-10 Julabo). Cold dissociation studies were carried out by incubating the enzymes for 20 h at 2°C at a concentration of 1 mg/ml in Tricine-KCl, 2 mM EDTA, 5 mM 2-mercaptoethanol and 50 μM PLP. The HPLC column was loaded with 20 μl of Trpase in Tricine-KCl. The HPLC-chromatogram showed two peaks. The first displayed a PLP-bound characteristic absorption spectrum of the tetrameric form with maxima at 278, 337 and 420 nm, and the second displayed a PLP-free enzyme peak, which is the dimeric form, characterized by the absence of the 337 and 420 nm peaks. Control samples incubated at 25°C for 1 h fully preserved their tetrameric structure. All data of specific activity and degree of dissociation are presented with the associated standard errors. HPLC measurements of apo Trpases were performed in the same buffer in the absence of PLP. The HPLC chromatogram of apo Trpases revealed two peaks with maximum absorbance at 278 nm.
High pressure studies
The effect of hydrostatic pressure on the absorption spectra measured using a Cary 14 UV-VIS spectrophotometer modified by OLIS, Inc., containing a high-pressure cell from ISS (Champaign, IL) and equipped with a manual pressure pump. The cell was maintained at 25°C with an external circulating water bath. Samples (1.2 ml) containing 1 mg/ml Trpase in 0.1 M triethanolamine hydrochloride, pH 8.0, were enclosed in quartz bottles with a 9 mm path length immersed in spectroscopic ethanol as the pressurizing fluid.
The equilibrium constant can be related to the change in absorbance at a particular wavelength by Eq. 3 for simple two species equilibrium, where Ap is the absorbance at pressure P, Ao is the absorbance at 0 bar, and A∞ is the absorbance at infinite pressure. The values of Ao, A∞, Keq and ΔVo were obtained by global fitting of the spectra at all wavelengths from 300 to 550 nm throughout the entire range of pressures to Eq. 1 and 3 using the GlobalWorks program provided by OLIS, Inc. . The protein concentration would be expected to increase with pressure because of the compressibility of water, which would result in a maximum increase in concentration of 4.7% at 1200 bar. However, the spectra were not corrected for the pressure effect, and the experimental results nonetheless fit well to the model, with low standard errors. The data also show an isosbestic point in the spectra, indicating that the concentration change due to pressure does not significantly affect the results.
Crystallization of Y74F and C298S
Crystallization experiments were carried out at 20°C, and were set up using the hanging-drop vapor-diffusion method with siliconized cover-slips and Linbro 24-well tissue culture plates. Droplets ranging in size from 5 to 10 μl prepared by mixing equal volume of the protein solution and the reservoir solution and were equilibrated with 1.0 ml of reservoir solution at room temperature (20°C). The protein at concentration of 30-50 mg/ml in 50 mM Tris, 100 mM KCl pH 7.5, 2 mM EDTA and 5 mM 2-mercaptoethanol was mixed with the reservoir solution containing 30%(w/v) PEG 400, 100 mM HEPES pH 7.5, 200 mM MgCl2, 5 mM 2-mercaptoethanol .
Data collection and structure determination
Crystals were transferred to Paratone oil (Hampton Research) and excess liquid was removed, followed by freezing in liquid nitrogen. Diffraction data set for the apo Y74F mutant was collected using a Rigaku RU3H rotating anode generator and MAR345 image plate detector at 100 K. For the apo C298S the diffraction data set was collected at low temperature (100 K) using a Raixs IV electronic area detector and a Rigaku RU-200 HB generator. Unit cell parameters, crystal orientation and integration of reflection intensities were determined using the HKL2000 package . Refinement of the data was done using CNS  and manual corrections of the model were carried out with the graphics program O .
We gratefully thank Miss Marina de Leeuw for her most valuable contribution to the preparation of this manuscript. This work was partially support by the James Frank Foundation on - Light Matter Interaction, (to AHP), and by the Helen Soref and the Stanley Medical Research Foundations (to OA).
- Bock PE, Frieden C: Another Look at Cold Lability of Enzymes. Trends in Biochemical Sciences 1978, 3(5):100–103. 10.1016/S0968-0004(78)80013-9View ArticleGoogle Scholar
- Bock PE, Gilbert HR, Frieden C: Analysis of the cold lability behavior of rabbit muscle phosphofructokinase. Biochem Biophys Res Commun 1975, 66(2):564–569. 10.1016/0006-291X(75)90547-1View ArticlePubMedGoogle Scholar
- Velichko IV, Volk SE, Dudarenkov VY, Magretova NN, Chernyak VY, Goldman A, Cooperman BS, Lahti R, Baykov AA: Cold Lability of the Mutant Forms of Escherichia-Coli Inorganic Pyrophosphatase. FEBS Lett 1995, 359(1):20–22. 10.1016/0014-5793(95)00003-RView ArticlePubMedGoogle Scholar
- Irias JJ, Olmsted MR, Utter MF: Pyruvate Carboxylase. Reversible Inactivation by Cold. Biochemistry 1969, 8(12):5136–5148. 10.1021/bi00840a068View ArticlePubMedGoogle Scholar
- Briganti F, Fong WP, Auld DS, Vallee BL: Invitro Dissociation and Reassociation of Human Alcohol-Dehydrogenase Class-I Isozymes. Biochemistry 1989, 28(13):5374–5379. 10.1021/bi00439a009View ArticlePubMedGoogle Scholar
- Shukuya R, Schwert GW: Glutamic acid decarboxylase. III. The inactivation of the enzyme at low temperatures. J Biol Chem 1960, 235: 1658–1661.PubMedGoogle Scholar
- Burns RO, Demoss RD: Properties of tryptophanase from Escherichia coli. Biochim Biophys Acta 1962, 65: 233–244. 10.1016/0006-3002(62)91042-9View ArticlePubMedGoogle Scholar
- Demoss JA: Studies on the mechanism of the tryptophan synthetase reaction. Biochim Biophys Acta 1962, 62: 279–293. 10.1016/0006-3002(62)90041-0View ArticlePubMedGoogle Scholar
- Almog O, Kogan A, Leeuw M, Gdalevsky GY, Cohen-Luria R, Parola AH: Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41. Biopolymers 2008, 89(5):354–359. 10.1002/bip.20866View ArticlePubMedGoogle Scholar
- Privalov PL, Makhatadze GI: Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J Mol Biol 1990, 213(2):385–391. 10.1016/S0022-2836(05)80198-6View ArticlePubMedGoogle Scholar
- Erez T, Gdalevsky G, Torchinsky YM, Phillips RS, Parola AH: Cold inactivation and dissociation into dimers of Escherichia coli tryptophanase and its W330F mutant form. Biochim Biophys Acta 1998, 1384(2):365–372.View ArticlePubMedGoogle Scholar
- Erez T, Phillips RS, Parola AH: Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation. FEBS Lett 1998, 433(3):279–282. 10.1016/S0014-5793(98)00931-4View ArticlePubMedGoogle Scholar
- Kunugi S, Tanaka N: Cold denaturation of proteins under high pressure. Biochim Biophys Acta 2002, 1595(1–2):329–344.View ArticlePubMedGoogle Scholar
- King L, Weber G: Conformational drift of dissociated lactate dehydrogenases. Biochemistry 1986, 25(12):3632–3637. 10.1021/bi00360a023View ArticlePubMedGoogle Scholar
- King L, Weber G: Conformational drift and cryoinactivation of lactate dehydrogenase. Biochemistry 1986, 25(12):3637–3640. 10.1021/bi00360a024View ArticlePubMedGoogle Scholar
- Wolynes PG, Onuchic JN, Thirumalai D: Navigating the folding routes. Science 1995, 267(5204):1619–1620. 10.1126/science.7886447View ArticlePubMedGoogle Scholar
- Franks F: Protein destabilization at low temperatures. Adv Protein Chem 1995, 46: 105–139. full_textView ArticlePubMedGoogle Scholar
- Isupov MN, Antson AA, Dodson EJ, Dodson GG, Dementieva IS, Zakomirdina LN, Wilson KS, Dauter Z, Lebedev AA, Harutyunyan EH: Crystal structure of tryptophanase. J Mol Biol 1998, 276(3):603–623. 10.1006/jmbi.1997.1561View ArticlePubMedGoogle Scholar
- Hogberg-Raibaud A, Raibaud O, Goldberg ME: Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5'-phosphate and monovalent cations. J Biol Chem 1975, 250(9):3352–3358.PubMedGoogle Scholar
- Suelter CH, Snell EE: Monovalent cation activation of tryptophanase. J Biol Chem 1977, 252(6):1852–1857.PubMedGoogle Scholar
- Suelter CH, Wang J, Snell EE: Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5'-phosphates. Anal Biochem 1976, 76(l):221–232. 10.1016/0003-2697(76)90280-3View ArticlePubMedGoogle Scholar
- Toraya T, Nihira T, Fukui S: Essential Role of Monovalent Cations in the Firm Binding of Pyridoxal 5'-Phosphate to Tryptophanase and P-Tyrosinase. Eur J Biochem 1976, 69: 411–419. 10.1111/j.1432-1033.1976.tb10925.xView ArticleGoogle Scholar
- Phillips RS: The Mechanism of Tryptophan Indole-lyase: Insights from Pre-steady-state Kinetics and Substrate and Solvent Isotope Effects. J Am Chem Soc 1989, 111: 727–730. 10.1021/ja00184a051View ArticleGoogle Scholar
- Phillips RS, Bender SL, Brzovic P, Dunn MF: Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry. Biochemistry 1990, 29(37):8608–8614. 10.1021/bi00489a016View ArticlePubMedGoogle Scholar
- Phillips RS, Demidkina TV, Faleev NG: Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase. Biochim Biophys Acta 2003, 1647(1–2):167–172.View ArticlePubMedGoogle Scholar
- Phillips RS, Demidkina TV, Zakomirdina LN, Bruno S, Ronda L, Mozzarelli A: Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes. J Biol Chem 2002, 277(24):21592–21597. 10.1074/jbc.M200216200View ArticlePubMedGoogle Scholar
- Erez T, Gdalevsky GY, Hariharan C, Pines D, Pines E, Phillips RS, Cohen-Luria R, Parola AH: Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase? Biochim Biophys Acta 2002, 1594(2):335–340.View ArticlePubMedGoogle Scholar
- Ku SY, Yip P, Howell PL: Structure of Escherichia coli tryptophanase. Acta Crystallogr D Biol Crystallogr 2006, 62(Pt 7):814–823. 10.1107/S0907444906019895View ArticlePubMedGoogle Scholar
- Tsesin N, Kogan A, Gdalevsky GY, Himanen JP, Cohen-Luria R, Parola AH, Goldgur Y, Almog O: The structure of apo tryptophanase from Escherichia coli reveals a wide-open conformation. Acta Crystallogr D Biol Crystallogr 2007, 63(Pt 9):969–974. 10.1107/S0907444907036396View ArticlePubMedGoogle Scholar
- Black SD, Mould DR: Development of hydrophobicity parameters to analyze proteins which bear post- or cotranslational modifications. Anal Biochem 1991, 193(1):72–82. 10.1016/0003-2697(91)90045-UView ArticlePubMedGoogle Scholar
- Gekko K, Tamura Y, Ohmae E, Hayashi H, Kagamiyama H, Ueno H: A large compressibility change of protein induced by a single amino acid substitution. Protein Science 1996, 5(3):542–545.PubMed CentralView ArticlePubMedGoogle Scholar
- Saito M, Kono H, Morii H, Uedaira H, Tahirov TH, Ogata K, Sarai A: Cavity-filling mutations enhance protein stability by lowering the free energy of native state. Journal of Physical Chemistry B 2000, 104(15):3705–3711. 10.1021/jp991717fView ArticleGoogle Scholar
- Atta-Asafo-Adjei E, Daldal F: Size of the amino acid side chain at position 158 of cytochrome b is critical for an active cytochrome bc1 complex and for photosynthetic growth of Rhodobacter capsulatus. Proc Natl Acad Sci USA 1991, 88(2):492–496. 10.1073/pnas.88.2.492PubMed CentralView ArticlePubMedGoogle Scholar
- Kulikova VV, Zakomirdina LN, Dementieva IS, Phillips RS, Gollnick PD, Demidkina TV, Faleev NG: Tryptophanase from Proteus vulgaris: the conformational rearrangement in the active site, induced by the mutation of Tyrosine 72 to phenylalanine, and its mechanistic consequences. Biochim Biophys Acta 2006, 1764(4):750–757.View ArticlePubMedGoogle Scholar
- Phillips RS, McPhie P, Miles EW, Marchal S, Lange R: Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase. Arch Biochem Biophys 2008, 470(1):8–19. 10.1016/j.abb.2007.11.003View ArticlePubMedGoogle Scholar
- Phillips RS, Miles EW, Holtermann G, Goody RS: Hydrostatic pressure affects the conformational equilibrium of Salmonella typhimurium tryptophan synthase. Biochemistry 2005, 44(21):7921–7928. 10.1021/bi050056bView ArticlePubMedGoogle Scholar
- Frye KJ, Royer CA: Probing the contribution of internal cavities to the volume change of protein unfolding under pressure. Protein Sci 1998, 7(10):2217–2222. 10.1002/pro.5560071020PubMed CentralView ArticlePubMedGoogle Scholar
- Matsuo H, Suzuki Y, Sawamura S: Solubility of alpha-amino acids in water under high pressure: glycine, L-alanine, L-valine, L-leucine, and L-isoleucine. Fluid Phase Equilibria 2002, 200(2):227–237. 10.1016/S0378-3812(02)00029-8View ArticleGoogle Scholar
- Rother K, Preissner R, Goede A, Frommel C: Inhomogeneous molecular density: reference packing densities and distribution of cavities within proteins. Bioinformatics 2003, 19(16):2112–2121. 10.1093/bioinformatics/btg292View ArticlePubMedGoogle Scholar
- Otwinowski Z, Minor W: Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology 1997, 276: 307–326. full_textView ArticleGoogle Scholar
- Kamath AV, Yanofsky C: Characterization of the tryptophanase operon of Proteus vulgaris. Cloning, nucleotide sequence, amino acid homology, and in vitro synthesis of the leader peptide and regulatory analysis. J Biol Chem 1992, 267(28):19978–19985.PubMedGoogle Scholar
- Kirsch JF, Eichele G, Ford GC, Vincent MG, Jansonius JN, Gehring H, Christen P: Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J Mol Biol 1984, 174(3):497–525. 10.1016/0022-2836(84)90333-4View ArticlePubMedGoogle Scholar
- Phillips RS, Ravichandran K, Vontersch RL: Synthesis of L-Tyrosine from Phenol and S-(Ortho-Nitrophenyl)-L-Cysteine Catalyzed by Tyrosine Phenol-Lyase. Enzyme and Microbial Technology 1989, 11(2):80–83. 10.1016/0141-0229(89)90064-1View ArticleGoogle Scholar
- Phillips RS, Gollnick PD: Evidence that cysteine 298 is in the active site of tryptophan indole-lyase. J Biol Chem 1989, 264(18):10627–10632.PubMedGoogle Scholar
- Kogan A, Gdalevsky GY, Cohen-Luria R, Parola AH, Goldgur Y: Crystallization and preliminary X-ray analysis of the apo form of Escherichia coli tryptophanase. Acta Crystallogr D Biol Crystallogr 2004, 60(Pt 11):2073–2075. 10.1107/S0907444904022425View ArticlePubMedGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248–254. 10.1016/0003-2697(76)90527-3View ArticlePubMedGoogle Scholar
- Phillips RS, Gollnick P: The environments of Trp-248 and Trp-330 in tryptophan indole-lyase from Escherichia coli. FEBS Lett 1990, 268(1):213–216. 10.1016/0014-5793(90)81011-CView ArticlePubMedGoogle Scholar
- Suelter CH, Wang J, Snell EE: Direct spectrophotometric assay of tryptophanase. FEBS Lett 1976, 66(2):230–232. 10.1016/0014-5793(76)80510-8View ArticlePubMedGoogle Scholar
- Edwards RA, Jickling G, Turner RJ: The light-induced reactions of tryptophan with halocompounds. Photochem Photobiol 2002, 75(4):362–368. Publisher Full Text 10.1562/0031-8655(2002)075<0362:TLIROT>2.0.CO;2View ArticlePubMedGoogle Scholar
- Raibaud O, Goldberg ME: The tryptophanase from Escherichia coli K-12. II. Comparison of the thermal stabilities of apo-, holo-, and hybrid enzymes. J Biol Chem 1973, 248(10):3451–3455.PubMedGoogle Scholar
- Phillips RS, Holtermann G: Differential effects of temperature and hydrostatic pressure on the formation of quinonoid intermediates from L-Trp and L-Met by H463F mutant Escherichia coli tryptophan indole-lyase. Biochemistry 2005, 44(43):14289–14297. 10.1021/bi051062aView ArticlePubMedGoogle Scholar
- Matheson IBC, Desa RJ: Robust Multivariate-Analysis Applied to Separation of Components in Absorption-Spectra. Computers & Chemistry 1990, 14(2):157–164. 10.1016/0097-8485(90)80019-XView ArticleGoogle Scholar
- Brunger AT, Adams PD, Rice LM: Recent developments for the efficient crystallographic refinement of macromolecular structures. Curr Opin Struct Biol 1998, 8(5):606–611. 10.1016/S0959-440X(98)80152-8View ArticlePubMedGoogle Scholar
- Jones TA: A graphics model building and refinement system for macromolecules. J Appl Crystallogr 1978, 11: 268–272. 10.1107/S0021889878013308View ArticleGoogle Scholar
- Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982, 157(1):105–132. 10.1016/0022-2836(82)90515-0View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.